Histomorphometric comparison of the human, swine, and ovine collecting systems.

The ovine kidney has been recently determined to be a better model than the swine kidney for the study of collecting system healing after partial nephrectomy. However, there is no histological study comparing the collecting systems of these species. To compare human, swine, and ovine collecting systems using histomorphometry. The collecting systems of 10 kidneys from each species (human, swine, and ovine) were processed for histomorphometry. The thickness of the three layers (mucosal connective tissue, submucosal muscular, and adventitial connective tissue) were measured. The densities of smooth muscle fibers, elastic system fibers, and cells were also measured. Additionally, blood vessel density in the adventitial connective tissue was measured. Analysis of the collecting systems from the three species presented several differences. The adventitial connective tissue from the swine samples was thicker, with more blood vessels and smooth muscle fibers, compared with that from the human and ovine samples. Swine also had higher density of elastic fibers on the submucosal muscular layer. Ovine and human collecting systems shared several similar features, such as blood vessel and elastic fiber density in all layers and the density of cellular and muscular fibers in the submucosal muscular and adventitial connective tissue layers. The collecting system of the ovine kidney is more similar to that of the human kidney compared with that of the swine kidney. This may explain the differences between the healing mechanisms in swine and those in humans and sheep after partial nephrectomy. Anat Rec, 299:967-972, 2016. © 2016 Wiley Periodicals, Inc.

Renal involvement in leptospirosis: the effect of glycolipoprotein on renal water absorption.

BACKGROUND: Leptospirotic renal lesions frequently produce a polyuric form of acute kidney injury with a urinary concentration defect. Our study investigated a possible effect of the glycolipoprotein, (GLPc) extracted from L. interrogans, on vasopressin (Vp) action in the guinea pig inner medullary collecting duct (IMCD). METHODS: The osmotic water permeability (Pf µm/s) was measured by the microperfusion in vitro technique. AQP2 protein abundance was determined by Western Blot. Three groups were established for study as follows: Group I, IMCD from normal (ngp, n = 5) and from leptospirotic guinea-pigs (lgp-infected with L. interrogans serovar Copenhageni, GLPc, n = 5); Group II, IMCD from normal guinea-pigs in the presence of GLPc (GLPc group, n = 54); Group III, IMCD from injected animals with GLPc ip (n = 8). RESULTS: In Group I, PFS were: ngp--61.8±22.1 and lgp--8.8±12.4, p<0.01 and the urinary osmolalities were: lgp--735±64 mOsm/Kg and ngp--1,632±120 mOsm/Kg. The lgp BUN was higher (176±36 mg%) than the ngp (56±9 mg%). In Group II, the Pf was measured under GLPc (250 µg/ml) applied directly to the bath solution of the microperfused normal guinea-pig IMCDs. GLPc blocked Vp (200 pg/ml, n = 5) action, did not block cAMP (10(-4) M), and Forskolin (Fors--10(-9) M) action, but partially blocked Cholera Toxin (ChT--10(-9) M) action. GLP from L.biflexa serovar patoc (GLPp, non pathogenic, 250 µg) did not alter Vp action. In Group III, GLPc (250 µg) injected intraperitoneally produced a decrease of about 20% in IMCD Aquaporin 2 expression. CONCLUSION: The IMCD Pf decrease caused by GLP is evidence, at least in part, towards explaining the urinary concentrating incapacity observed in infected guinea-pigs.

Actin cytoskeletal and functional studies of the proximal convoluted tubules after preservation.

BACKGROUND: Proximal tubule cells have specialized apical membranes with microvilli that provide an extensive surface area for unidirectional transport of solute from lumen to blood. The major structural solute component is F-actin, which interacts with transmembrane proteins, including ion transport molecules related to normal absorptive and secretory functions. Our study was to evaluate F-actin and fluid absorption (Jv) in proximal tubules after exposure to preservation solutions. METHODS: In vitro microperfusion technique and immunohistochemistry analysis. RESULTS: 1. Absorptions were similar in 1- and 24-hour-preserved tubules, as well as in fresh tubules. The exception was tubules for 24 hours in Euro-Collins solution, which did not show absorption, suggesting that it was affected. 2. Fluorescence intensity of actin tubules preserved for 1 hour in both solutions showed similar values to each other and to the control group; tubules preserved for 24 hours in both solutions were similar to each other, although statistically different than the control group and those preserved for 1 hour in Belzer (UW) solution. CONCLUSION: There were differences among groups in the distribution of F-actin; Jv values were different for 24-hour preservation in each solution, whereas fluorescence intensity was similar in both 24-hour solutions. Thus, actin cytoskeleton was not responsible for it, because 24-hour preservation in UW showed Jv results comparable to the control group.

Differential distribution of the vasopressin V receptor along the rat nephron during renal ontogeny and maturation.

BACKGROUND: Ontogeny and cellular distribution of vasopressin receptors in the kidney are key factors determining the role of vasopressin in renal physiology. Expression of vasopressin V(2) receptor (V(2)R) mRNA and the immunoreactive protein in rat kidney were investigated. METHODS: An antiserum directed to epitope TLD25 of the rat V(2)R sequence was characterized by Western blotting. Expression of V(2)R mRNA was assessed by reverse transcription-polymerase chain reaction (RT-PCR), and on protein level by immunohistochemistry. RESULTS: Specificity of the antiserum was documented by Western blots from cells expressing a fusion protein of V(2)R and GFP. Using lysates of rat kidney and of native cell lines expressing V(2)R but not V(1)R, our antiserum to peptide TLD25 revealed a major band of 55 kD corresponding to the monomeric form of V(2)R, and a band of 110 kD most likely representing the homodimeric form of the receptor. This highly specific antiserum allowed us to localize the V(2)R in thick ascending limbs, distal convoluted and connecting tubules, and in collecting ducts. During ontogeny, immunoreactivity was first observed at the luminal membrane on prenatal day 20, emerging at the basolateral side from postnatal day 5 on. RT-PCR demonstrated V(2)R transcripts from prenatal day 18 to gradually increasing thereafter. CONCLUSION: Expression of V(2)R is first detectable in the late embryonic stage of rat ontogeny starting from day E18 and gradually increasing with kidney maturation. In the adult kidney, V(2)R is differentially distributed in the various nephron segments.

Differential role of Na+/H+ exchange isoforms NHE-1 and NHE-2 in a rat cortical collecting duct cell line.

The Na+/H+ exchanger (NHE) constitutes a gene family containing several isoforms that display different membrane localization and are involved in specialized functions. Although basolateral NHE-1 activity was described in the cortical collecting duct (CCD), the localization and function of other NHE isoforms is not yet clear, This study examines the expression, localization, and regulation of NHE isoforms in a rat cortical collecting duct cell line (RCCD1) that has previously been shown to be a good model of CCD cells. Present studies demonstrate the presence of NHE-1 and NHE-2 isoforms, but not NHE-3 and NHE-4, in RCCD1 cells. Cell monolayers, grown on permeable filters, were placed on special holders allowing independent access to apical and basolateral compartments. Intracellular pH (pHi) regulation was spectrofluorometrically studied in basal conditions and after stimulation by NH4Cl acid load or by a hyperosmotic shock. In order to differentiate the roles of NHE-1 and NHE-2, we have used HOE-694, an inhibitor more selective for NHE-1 than for NHE-2. The results obtained strongly suggest that NHE-1 and NHE-2 are expressed in the basolateral membrane but that they have different roles: NHE-1 is responsible for pHi recovery after an acid load and NHE-2 is mainly involved in steady-state pHi and cell volume regulation.

Nifedipine-activated Ca(2+) permeability in newborn rat cortical collecting duct cells in primary culture.

To characterize Ca(2+) transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca(2+) concentration ([Ca(2+)](i)) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 microM) produced an increase in [Ca(2+)](i) from 87.6 +/- 3.3 nM to 389.9 +/- 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca(2+)](i) in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca(2+)](i). Experiments in the presence of EGTA showed that external Ca(2+) was required for the nifedipine effect, while lanthanum (20 microM), gadolinium (100 microM), and diltiazem (20 microM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K(+) channels were not involved in the nifedipine-induced [Ca(2+)](i) rise. H(2)O(2) also triggered [Ca(2+)](i) rise. However, nifedipine-induced [Ca(2+)](i) increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca(2+) transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca(2+) channel of capacitive type (either transient receptor potential or leak channel).

Vasopressin regulates water flow in a rat cortical collecting duct cell line not containing known aquaporins.

Transepithelial water movements and arginine-vasopressin (AVP)-associated ones were studied in a renal cell line established from a rat cortical collecting duct (RCCD(1)). Transepithelial net water fluxes (J(w)) were recorded every minute in RCCD(1) monolayers cultured on permeable supports. Spontaneous net water secretion was observed, which was inhibited by serosal bumetanide (10(-5) m), apical glibenclamide (10(-4) m) and apical BaCl(2) (5 x 10(-3) m). RT-PCR, RNAse protection and/or immunoblotting experiments demonstrated that known renal aquaporins (AQP1, AQP2, AQP3, AQP4, AQP6 and AQP7) were not expressed in RCCD(1) cells. AVP stimulates cAMP production and sodium reabsorption in RCCD(1) cells. We have now observed that AVP significantly reduces the spontaneous water secretory flux. The amiloride-sensitive AVP-induced increase in short-circuit current (I(sc)) was paralleled by a simultaneous modification of the observed J(w): both responses had similar time courses and half-times (about 4 min). On the other hand, AVP did not modify the osmotically driven J(w) induced by serosal hypertonicity. We can conclude that: (i) transepithelial J(w) occurs in RCCD(1) cells in the absence of known renal aquaporins; (ii) the "water secretory component" observed could be linked to Cl- and K = secretion; (iii) the natriferic response to AVP, preserved in RCCD(1) cells, was associated with a change in net water flux, which was even observed in absence of AQP2, AQP3 or AQP4 and (iv) the hydro-osmotic response to AVP was completely lost.

Micropuncture study on urea movements in the kidney cortical tubules of low protein fed sheep.

Micropuncture studies of late proximal, early and late distal cortical tubules were carried out on kidneys of normal (NP) and low (LP) protein fed sheep in order to investigate the participation of these segments in the urea sparing induced by protein restriction in the diet. A low protein diet induced significant reductions in the fractional (-54%) and total (-84%) urea excretion, revealing an enhanced capacity for urea conservation. Micropuncture data did not show any difference in the proximal tubule functions between both groups of sheep. In distal cortical tubules the fractional delivery of urea (early distal, 0.61 +/- 0.06 for NP and 0.77 +/- 0.06 for LP sheep, not significant (NS); late distal, 0.45 +/- 0.07 for NP and 0.71 +/- 0.09 for LP sheep, P < 0.05) showed a relatively larger amount of urea present in the late distal tubule of protein restricted sheep. The tubular fluid-to-plasma inulin ratio in the late distal tubule was found to be lower in LP sheep (4.33 +/- 0.23 versus 8.58 +/- 0.9 in NP sheep, P < 0.01). The tubular flow rate, reduced in the early distal tubules of LP sheep (10.87 +/- 0.99 versus 18.92 +/- 2.58 nL.min-1 in NP sheep, P < 0.05), was not different in the late distal tubules from values in normally fed animals (6.65 +/- 0.90 versus 7.73 +/- 0.94 nL.min-1 in NP sheep, NS). These findings suggest a decreased distal water reabsorption coincident with the relatively larger amounts of intraluminal urea in LP sheep. This relatively larger urea delivery to the initial collecting duct could increase the subsequent urea reabsorption in protein restricted sheep.

Effect of peritubular hypertonicity on water and urea transport of inner medullary collecting duct.

The effect of bath fluid hypertonicity on hydraulic conductivity (Lp) and [14C]urea permeability (Pu) of the distal inner medullary collecting duct (IMCD) was studied in the absence and in the presence of vasopressin (VP) using the in vitro microperfusion technique of rat IMCD. In the first three groups of IMCD, we observed that in the absence of VP the Lp was not different from zero when the osmotic gradient was created by hypotonic perfusate and isotonic bath fluid, but it was significantly greater than 1.0 x 10(-6) cm.atm-1.s-1 when the osmotic gradient was created by hypertonic bath and isotonic perfusion fluid. The increase in Lp was observed when the hypertonicity of the bath fluid was produced by the addition of NaCl or raffinose, but no such effect was observed with urea. The stimulated effect of bath fluid hypertonicity on Lp was also observed in the IMCD obtained from Brattleboro homozygous rats in which VP is absent. The NaCl hypertonic bath increased the Pu in the absence of VP. In another series of experiments with VP (10(-10) M) we observed that the hypertonic bath fluid increased in a reversible manner the VP-stimulated Lp of distal IMCD. However, the NaCl hypertonicity of the bath fluid was not able to increase dibutyryladenosine 3',5'-cyclic monophosphate-stimulated Lp. The Pu stimulated by VP (10(-10) M) increased twofold when the bath fluid was hypertonic. Therefore hypertonicity of the peritubular fluid produced by the addition of NaCl or raffinose increases the Lp and Pu in the absence and in the presence of VP. No such effect was noted with the addition of urea.

Effect of atrial natriuretic factor and cyclic guanosine monophosphate on water and urea transport in the inner medullary collecting duct.

We examined the action of high (2 x 10(-8)M) and low (6 x 10(-9)M) concentrations of atrial natriuretic factor (ANF) on water and urea transport in the rat inner medullary collecting duct (IMCD) using the in vitro microperfusion technique. We measured the hydraulic conductivity (Lp x 10(-6) cm/atm per second) and both lumen-to-bath (Pu(lb] and bath-to-lumen (Pu(bl)) 14C-urea permeabilities (Pu x 10(-5) cm/s) in the absence and in the presence of vasopressin (VP). High concentrations of ANF were able to inhibit the maximum activity of (50 microU/ml) VP-stimulated Lp but physiological concentration of ANF inhibit only submaximum activity (10 microU/ml) of VP-stimulated Lp. The hydrosomotic effect of dibutyryl-cyclic 3.5 adenosine monophosphate (cAMP) (10(-4)M) was unchanged by high concentrations of ANF (2 x 10(-8)M). Also we found that high (10(-4)M) and low (10(-6)M) concentrations of exogenous cyclic 3,5-guanosine monophosphate (GMP) while unable to change the Lp in the absence of VP, decreased the maximum activity of VP-stimulated Lp significantly. We also found that ANF inhibits partially and in a reversible manner the VP-stimulated Pu(lg) but not the VP-stimulated Pu(bl). These results demonstrated that plasma concentrations of ANF observed during volume expansion (10(-10)M) are able to inhibit submaximum activity of VP-stimulated (10 microU/ml) Lp in the rat IMCD, this effect seems to occur before cAMP formation and it appears to be mediated by cGMP. ANF (6 x 10(-9)M) also reduced the VP-stimulated urea outflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Channels for water flow in epithelia: characteristics and regulation.

Pos and Pd, osmotic and diffusive water permeability coefficients of isolated rabbit proximal tubule (PT) cells (expressed per cm2 of real cell membrane area, in microns/sec) are: Pos 396; Pd, 22; Pos/Pd, 18 (control), and after exposure to parachloromercuribenzenesulfonate (pCMBS): Pos, 32; Pd, 10; Pos/Pd, 3. The sulfhydryl reagent dithiothreitol (DTT) reverts pCMBS action. The activation energies (kcal/mol) are Pos, 3.2 (control); 9.2 (pCMBS); Pd, 5.2 (control) and 9.1 (pCMBS). Thus water channels pierce the control plasma membrane and are reversibly closed by pCMBS. High control PT permeabilities are comparable with those of amphibian urinary bladder and collecting tubules (CT) stimulated with antidiuretic hormone (ADH), and low PT (pCMBS) values with those of CT in the resting state, respectively. Transcellular permeability is regulated in PT by the state of sulfhydryl groups and in CT by ADH induced insertion (or, no ADH, suppression) of water channels. In PT (a) large extracellular markers are dragged by water flow indicating extracellular solute-water interaction, (b) transepithelial Pos is much higher than transcellular Pos. Therefore water also flows paracellularly, in addition to the transcellular flow. In PT paracellular permeability is increased if urea in lumen is higher than in blood. It is reduced in the reverse situation. In CT paracellular permeability is virtually zero (resting condition). It may be increased by high lumen urea. Thus, paracellular permeability (which is significant in control PT and zero in CT) can be regulated by changes in the transepithelial urea concentrations. Transcellular permeability depends on the number of channels/cm2 epithelium, their probability of being opened and their individual permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Channels for water flow in epithelia: characteristics and regulation

Pos y Pd, los coeficientes de permeabilidad osmótica y difusiva al agua, de túbulos proximales (TP) de conejo (por cm**2 de membrana celular real, micronm/s) son Pos, 396; Pd, 22; Pos/Pd, 18 (controles). Con paracloromercuribencenosulfonato son Pos, 32; Pd, 10; Pos/Pd, 3. El reactivo de grupos sulfhidrilos ditiotreitol (DTT) revierte la acción del pCMBS. Las energías de activación (Kcal/mol) son Pos, 3.2 (controles); 9.2 (pCMBS); Pd, 5.2 (controles, 9.1 (pCMBS). Por lo tanto, canales acuosos atraviesan la membrana celular control y son cerrados por pCMBS. Las altas permeabilidades de TP (controles) son similares a las de la vejiga urinaria de anfibio (un análogo del túbulo colector, TC), estimulada con hormona antidiurética (ADH) y los valores bajos con pCMBS en TP recuerdan los de TC en reposo (sin ADH). La permeabilidad transcelular puede ser regulada por el estado de grupos sulfhidrilo en TP y por la adición de canales acuosos por la HAD (o su supresión, reposo). En T.P.(a) no-electrólitos extracelulares son arrastrados por el flujo de agua indicando interacción extracelular aguasoluto; (b) la Pos transepitelial es mucho mayor que la transcelualr. Por consiguiente, en adición al flujo transcelular hay flujo paracelular de agua. La permeabilidad en TP se incrementa si la urea luminal es mayor que la sanguínea (en 15-50 mM) y se reduce en la situación inversa. En TD (control) la permeabilidad paracelular es cero. Se incrementa con urea en condición control en TP y muy pequeña en TC...

Channels for water flow in epithelia: characteristics and regulation

Pos y Pd, los coeficientes de permeabilidad osmótica y difusiva al agua, de túbulos proximales (TP) de conejo (por cm**2 de membrana celular real, micronm/s) son Pos, 396; Pd, 22; Pos/Pd, 18 (controles). Con paracloromercuribencenosulfonato son Pos, 32; Pd, 10; Pos/Pd, 3. El reactivo de grupos sulfhidrilos ditiotreitol (DTT) revierte la acción del pCMBS. Las energías de activación (Kcal/mol) son Pos, 3.2 (controles); 9.2 (pCMBS); Pd, 5.2 (controles, 9.1 (pCMBS). Por lo tanto, canales acuosos atraviesan la membrana celular control y son cerrados por pCMBS. Las altas permeabilidades de TP (controles) son similares a las de la vejiga urinaria de anfibio (un análogo del túbulo colector, TC), estimulada con hormona antidiurética (ADH) y los valores bajos con pCMBS en TP recuerdan los de TC en reposo (sin ADH). La permeabilidad transcelular puede ser regulada por el estado de grupos sulfhidrilo en TP y por la adición de canales acuosos por la HAD (o su supresión, reposo). En T.P.(a) no-electrólitos extracelulares son arrastrados por el flujo de agua indicando interacción extracelular aguasoluto; (b) la Pos transepitelial es mucho mayor que la transcelualr. Por consiguiente, en adición al flujo transcelular hay flujo paracelular de agua. La permeabilidad en TP se incrementa si la urea luminal es mayor que la sanguínea (en 15-50 mM) y se reduce en la situación inversa. En TD (control) la permeabilidad paracelular es cero. Se incrementa con urea en condición control en TP y muy pequeña en TC... (AU)